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N-Nitrosodimethylamine (NDMA), but not N-nitroso-N-methylurea (MNU) was more mutagenic in the Salmonella hisG428 strain, TA104, than in the hisG46 strain, TA100 in the presence of rat or hamster liver S-9 mix. As both NMDA and MNU can give rise to methyldiazonium ion (MDI) it appears that NDMA can be metabolized to an additional mutagen with a higher activity in TA104. The effects of UV and error-prone repair on NDMA and MNU-induced mutagenesis in TA104 were also different. alpha-Acetoxy-NDMA, which gives rise to the NDMA metabolite, alpha-hydroxy-NDMA, was more mutagenic in TA104 than TA100, under certain conditions. Several metabolites of NDMA (formaldehyde, 1,1-dimethylhydrazine and nitrite) were not significantly mutagenic at the concentrations that could have been generated from NDMA. It was previously reported that the microsomal-mediated mutagenesis induced by NDMA is greatly increased by cytosol in TA104, but not in TA100. The current study found that when cytosol was separated into a high and a low mol. wt fraction, neither greatly enhanced microsomal-mediated mutagenesis by NDMA in TA104. Addition of NAD to the high, but not the low mol. wt fraction resulted in greatly enhanced activation of NDMA to a mutagen in TA104. The enhancement by cytosol of NDMA-induced mutagenesis in hisG428 was only observed when both microsomes and cytosol were simultaneously present. These observations indicate that (i) the precursor to the ultimate mutagen is relatively short-lived; and (ii) the metabolism of alpha-hydroxy-NDMA to a secondary mutagenic metabolite, possibly N-nitroso-N-methylformamide, by alcohol dehydrogenase may be responsible for the ultimate mutagen with relatively high activity in TA104.

To investigate the influence of different types of metabolic activation (9,000 x g supernatant (S9) activation vs. a host-mediated approach) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutational specificity, we determined by DNA sequencing the distribution of forward mutations recovered in the N-terminal region of the lac1 gene of Escherichia coli. After activation with the S9 liver fraction from rats treated with Aroclor 1254, a diverse spectrum of mutations was recovered, with 55% of the events being G:C-->A:T transitions. In contrast, after the host-mediated assay in mice, G:C-->A:T transitions accounted for over 94% of the mutations recovered. Generally, NNK metabolism can proceed through two distinct pathways, involving either alpha-methyl or methylene hydroxylation. These two pathways produce different distributions of DNA damage. The difference in the mutational spectra we observed thus likely reflects the difference in the contributions of each pathway under the two different treatment conditions.

The mutational activities and specificities of several N-nitrosamines in Salmonella recovered from mouse liver in the host-mediated assay (HMA) were compared with the specificities of related direct-acting N-nitroso compounds in vitro. The specificities of the direct-acting methyl, ethyl, propyl, and 2-hydroxypropyl compounds were all different and presumably are attributable to the DNA adducts resulting from the corresponding alkyldiazonium or carbonium ions. Introduction of a 2-hydroxyl group greatly influenced the mutational specificity. The 2-oxopropyl compound showed the same specificity as the methyl compound. This result is consistent with one of the known breakdown pathways of the oxopropyl diazonium ion (or related reactive species), which leads to a methyl diazonium ion. The N-nitrosodialkylnitrosamines N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosodipropylamine (NDPA), which all require metabolic activation, showed specificities in the HMA similar to those of their direct-acting counterparts. The cyclic nitrosamine N-nitrosopyrrolidine was weakly active in the HMA, although its direct-acting derivative was a potent mutagen in vitro. The results for NDMA and NDEA were consistent with most previous studies of the metabolism of these compounds in vivo. However, NDPA can yield methylating, and probably hydroxypropylating, species in addition to propyldiazonium ion. As the specificity of NDPA was similar to that of a propylating agent, NDPA appears to lead to genotoxic products in the mouse liver mainly by direct alpha-hydroxylation. The initial results described here indicate that mutational specificity in the HMA can be used to deduce metabolic pathways leading to genotoxic products when the appropriate proximate mutagens are available as standards. Furthermore, we observed a reasonable correlation between potency in the HMA and hepatocarcinogenesis.

The relationships between DNA alkylation, DNA repair and mutagenesis by N-nitroso compounds in Salmonella were examined. DNA adducts formed by treatment of the bacteria with N-nitroso compounds were monitored. Critical to the study was establishing which adducts led to mutations. Two methods were employed. In one, correlations in the dose-responses for adducts and mutagenesis were sought. For instance O6-methyl- and -ethyl-guanine, in contrast to other adducts, exhibited thresholds in their accumulation in Salmonella DNA, and mutagenesis at GC base pairs also exhibited the same threshold, suggesting a dependence of mutagenesis on the O6-alkylguanines. In the second method, mutagenesis induced by different mutagens with overlapping adduct spectra was compared. For example, EMS and ENU generate similar ratios of adenine adducts, but only ENU produces thymine adducts, and only ENU induced AT-GC and AT-CG base changes. These observations suggested that ethylthymines led to these mutations. Furthermore, it was found that these mutations were largely dependent on the presence of the plasmid, pKM101, indicating that error-prone repair activity contributes importantly in their processing to mutations. When DNA adducts by N-nitrosopyrrolidine were examined it was found that only one major adduct was detected in an excision-repair-deficient strain, and that this adduct was not present in a repair-proficient strain. Mutagenesis was also greatly reduced in the proficient strain, suggesting that mutagenesis was dependent on this adduct. From the relationships between premutagenic adduct levels and mutagenesis it was possible to calculate estimated values for the mutational efficiencies for several adducts. This calculation assumed an average distribution of adducts and mutations and required knowledge of the target size and the types of mutations that could lead to phenotypic changes. For the unrepaired O6-methyl- and -ethyl-guanines, and the O-ethylthymines the mutational efficiencies were high (ca. 30-70%), but for the N-nitrosopyrrolidine adduct it was low (ca. 1%). Initial studies were carried out on the mutational specificities of two higher homologue N-nitroso compounds (the N-nitroso-N-propyl- and N-butyl-nitroguanidines) in uvrB/pKM101 strains. This class of nitroso compounds is known to form similar DNA adducts as ENU. Their specificities were similar to that of N-nitroso-N-ethylurea at a high dose except the fraction of mutations at AT base pairs was reduced. The fraction of GC-CG transversions was although low, increased. The mutational specificities of N-nitroso-N-methylurea and N-nitrosopyrrolidine were significantly different from the specificity of E

Microsomal-mediated mutagenesis induced by N-nitrosodimethylamine (NDMA) in Salmonella TA100 at neutral pH was only slightly affected by cytosol and was similar in its threshold type dose-response curve to mutagenesis induced by direct-acting N-nitroso-N-methyl compounds. However, mutagenesis in strain TA104 was greatly enhanced by cytosol and this mutagenesis did not exhibit a threshold. In the presence of microsomes alone NDMA was more potent in TA100 than TA104, but in the presence of microsomes plus cytosol (S-9 fraction) this order was reversed at the doses tested. A possible explanation for these results is that NDMA is metabolized by microsomes to a mutagen (presumably methyldiazonium ion; MDI) that is more potent in TA100 than in TA104, but in the presence of S-9 fraction a fraction of the NDMA is metabolized by a pathway leading to a different mutagen with a different specificity. The ratio of metabolism via these pathways appears to be dependent on pH.

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.

Little is known about the nature and possible genotoxic effects of the DNA adducts formed by N-nitrosopyrrolidine (NPYR) in whole animals. DNA binding in DNA isolated from [2,5-14C]NPYR-treated Salmonella was studied and attempts were made to monitor DNA adducts and correlate DNA binding with mutagenesis. NPYR was metabolized by hamster liver S-9 fraction in the presence of S.typhimurium TA1535 (uvrB-) or TA1975(uvrB+). DNA isolated from TA1535 contained about three times as much radioactivity as that isolated from TA1975, and NPYR-induced mutagenesis was several-fold higher in TA1535. The fraction of radioactivity incorporated into TA1535 was approximately 10(-5). Thermal hydrolysis of the 14C-containing DNA at neutral pH, followed by precipitation, released approximately 2/3 of the radioactivity into the supernatant. HPLC analysis of the supernatant revealed one major peak. This peak was absent in DNA from TA1975. Acid hydrolysis of the DNA precipitate after neutral hydrolysis released most of the residual radioactivity. Several small peaks were observed after HPLC analysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate. These results demonstrate that NPYR is capable of binding to Salmonella DNA yielding one major product after hydrolysis and this DNA binding product appears to be repaired by the excision repair system. The fact that the major peak of radioactivity released from Salmonella is only found in the strain which is efficiently reverted by NPYR suggests that mutagenesis is dependent on the DNA modification leading to this peak.

The DNA adducts and mutational profile produced by N-nitroso-N-ethylurea (ENU) in Salmonella are examined. The adduct profile produced by ENU in isolated DNA and at two doses in Salmonella were similar, with one exception: O6-ethylguanine (O6-EtG) was not detected at the low dose in Salmonella. This adduct was presumably repaired by a constitutive repair system. The premutagenic adducts, O2-ethyl-thymidine (O2-EtdT) and O4-ethylthymidine (O4-EtdT) were detected, with the former adduct present at higher levels. The mutational profile was also determined at the same doses by utilizing a system involving a series of histidine auxotrophs of Salmonella with differing mutagenic specificities and a further subclassification of the revertants. Four different patterns of mutagenesis were observed; these were dependent on dose and on the presence or absence of the plasmid pKM101. The mutational spectrum produced at the higher dose in strains without the plasmid consisted mainly of GC----AT transitions. At the high dose, in strains harboring pKM101, three base changes contributed importantly to the mutational spectrum: GC----AT, AT----GC, and AT----CG. At the low dose in the strains without pKM101, little mutagenesis was observed, and in strains containing pKM101, mutagenesis was greatly enhanced with the most frequent mutations resulting from AT----GC and AT----CG base changes. O6-EtG was presumably responsible for the bulk of the GC----AT transitions at the high dose. Calculations and evidence are presented indicating that O2-EtdT is responsible for at least some of the mutagenesis that occurs at AT base pairs. O4-EtdT and O2-EtdT are probably responsible for a major fraction of the AT----GC transitions, and we suggest that error-prone repair activity acting on O2-EtdT and/or O4-EtdT results in the AT----CG transversions.